Optoinjection methods

Abstract

Optoinjection method for transiently permeabilizing a target cell by (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.

1 . A method of rapidly introducing an exogenous molecule into a live population of cells, comprising the steps of: 
 providing a live population of cells in the presence of said exogenous molecule;    providing an energy source configured to deliver a beam of radiation;    positioning a container holding said live population of cells within the focus of said beam of radiation; and    contacting one or more cells of said live population of cells with said beam of radiation, wherein said beam of radiation has a diameter of at least 2 μm at the point of contact with said one or more cells;    wherein one or more cells of said live population of cells is transiently permeabilized to allow said exogenous molecule to enter.    
   
   
       2 . The method of  claim 1 , wherein said diameter of said beam of radiation is at least 5 μm at said point of contact with said live population of cells.  
   
   
       3 . The method of claims  1 , wherein said beam of radiation delivers at most 2, 1.5, 1, 0.7, 0.5, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01 or 0.005 μJ/μm 2 .  
   
   
       4 . The method of claims  1 , wherein said beam of radiation is delivered through a lens with a numerical aperture of at most 0.5, 0.4, 0.3, or 0.25.  
   
   
       5 . A method of rapidly introducing exogenous molecules into a live population of cells, comprising the steps of: 
 providing a live population of cells in the presence of exogenous molecules;    providing an energy source configured to deliver a beam of radiation;    positioning a container holding said live population of cells within the focus of said beam of radiation; and    contacting one or more cells of said live population of cells with said beam of radiation, wherein said beam of radiation delivers at most 2 μJ/μm 2 ;    wherein one or more cells of said live population of cells is transiently permeabilized to allow said exogenous molecules to enter.    
   
   
       6 . The method of  claim 5 , wherein said beam of radiation delivers a value selected from the group of at most 1.5, 1, 0.7, 0.5, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01 or 0.005 μJ/μm 2 .  
   
   
       7 . The method of claims  5 , wherein said beam of radiation is delivered through a lens with a numerical aperture of at most 0.5, 0.4, 0.3, or 0.25.  
   
   
       8 . The method of claims  6 , wherein said lens has an F-theta flat-field correction.  
   
   
       9 . A method of rapidly introducing exogenous molecules into a live population of cells, comprising the steps of: 
 providing a live population of cells in the presence of exogenous molecules;    providing an energy source configured to deliver a beam of radiation;    positioning a container holding said live population of cells within the focus of said beam of radiation; and    contacting one or more cells of said live population of cells with said beam of radiation, wherein said beam of radiation is delivered through a lens with a numerical aperture of at most 0.5;    wherein one or more cells of said live population of cells is transiently permeabilized to allow said exogenous molecules to enter.    
   
   
       10 . The method of  claim 9 , wherein said lens has a numerical aperture selected from the group of at most 0.4, 0.3, or 0.25.  
   
   
       11 . The method of claims  9 , wherein positioning said container holding said live population of cells within said focus of said beam of radiation further comprises the steps of: 
 illuminating a field-of-view containing said live population of cells;    analyzing an image of said live population of cells within said field-of-view; and    adjusting the distance between said population of cells and said lens.    
   
   
       12 . The method of claims  9 , wherein contacting said live population of cells with said beam of radiation comprises: 
 pulsing said energy source; and    steering the pulsed beam of radiation from said energy source to multiple spots within said live population of cells.    
   
   
       13 . The method of  claim 11 , wherein said pulsed beam of radiation is wider than any individual cell and is directed to the general area of said live population of cells.  
   
   
       14 . The method of claims  9 , wherein said lens comprises a working distance of at least 5, 7, or 10 mm.  
   
   
       15 . A method of rapidly introducing an exogenous molecule into a live cell, comprising: 
 providing a live cell in the presence of said exogenous molecule;    providing an energy source configured to deliver a beam of radiation; and    contacting said live cell with said beam of radiation, wherein said live cell is transiently permeabilized to allow said exogenous molecule to enter; and    wherein (a) said beam of radiation has a diameter of at least 2 μm at the point of contact with said live cell, (b) said beam of radiation delivers at most 2 μJ/μm 2 , (c) said beam of radiation is delivered through a lens with a numerical aperture of at most 0.5, or (d) two or more of (a), (b) or (c).    
   
   
       16 . The method of  claim 16 , wherein said beam of radiation has a diameter of at least 2, 5, 7, 10, 15, 20, 25, or 30 μm at the point of contact with said live cell.  
   
   
       17 . The method of  claim 15 , wherein said beam of radiation is wider than the cell or is directed to the general area of said cell.  
   
   
       18 . The method of  claim 15 , wherein two or more of the following apply (a) said beam of radiation has a diameter of at least 2 μm at the point of contact with said live cell, (b) said beam of radiation delivers at most 2 μJ/μm 2 , or (c) said beam of radiation is delivered through a lens with a numerical aperture of at most 0.5.  
   
   
       19 . The method of  claim 18 , wherein said beam of radiation has a diameter of at least 10 μm at the point of contact with said live cell.  
   
   
       20 . The method of  claim 18 , wherein (a), (b) and (c) apply.